KR20150043126A - A composition comprising Treculia africana extracts having anti-cancer activity - Google Patents

Abstract

The present invention relates to a pharmaceutical composition for preventing or treating colon cancer and to a food composition for preventing or alleviating colon cancer, wherein a Treculia africana extract is included as an active ingredient. The composition including the Treculia Africana extract of the present invention does not exhibit toxicity for normal cells but induces the apoptosis of colon cancer cells to exhibit a remarkably excellent colon cancer cell killing effect. Accordingly, the composition can be usefully applied as an anticancer composition so as to be used as the pharmaceutical composition or the food composition for preventing or treating colon cancer.

Description

[0001] The present invention relates to an anticancer composition comprising Treculia africana extract or extract as an active ingredient.

The present invention relates to a pharmaceutical composition for preventing or treating colorectal cancer comprising extract of Treculia africana as an active ingredient, and a food composition for preventing or ameliorating colon cancer.

Cancer is one of the major causes of death in Korea. There have been many studies to conquer cancer. However, it is an incurable disease that can not be regulated. In cancer, cells divide and grow uncontrollably to form malignant tumors and invade adjacent tissues of the body. In addition, cancer can migrate farther through lymphatic or blood flow as well as metastasis to adjacent tissues. Existing therapies for cancer include surgery, chemotherapy, and radiation therapy. Among these, chemotherapy using chemotherapeutic agents is now widely used for cancer treatment and is one of relatively well established treatment methods.

The term 'anticancer agent' refers to chemotherapeutic agents other than surgery and radiation therapy among the methods used to treat malignant tumors, and most of them are substances that inhibit the synthesis of nucleic acids and exhibit anticancer activity. Chemotherapeutic agents are largely classified into antimetabolites, alkylating agents, antimitotic drugs, and hormones. Methotrexate, 6-mercaptopurine, 6-thioguanine, 5-fluorouracil, and Cytarabine are examples of metabolic antagonists that inhibit the metabolic processes required for cancer cell proliferation. Examples of the alkylating agent that introduces an alkyl group into the DNA guanine to modify the DNA structure and cleave the chain to exhibit an anticancer effect include chlorambucil, cyclophosphamide, ethylenimine compound, thiotepa, (busulfan), a nitrosourea compound (carmustine), and a triazine compound (dacarbazine). As mitotic timing specificity drugs, mitotic inhibitors that inhibit mitosis by inhibiting mitosis by inhibiting mitosis include anticancer drugs such as actinomycin D, doxorubicin, bleomycin, mitomycin, vincristine, vinblastine Plant alkaloids, taxoids that are mitotic inhibitors including taxane rings, and the like. In addition, hormones such as corticosteroids or progesterone and platinum-containing compounds such as cisplatin are used as anticancer drugs.

Of these cancers, drug therapy is being attempted in the same way as the above-mentioned anticancer drugs for colon cancer, which is recognized as "cancer of advanced countries", occupying the second largest cancer in the Western world and the fourth largest cancer in the country . Typically, the antineoplastic compound 5-FU (5-fluorouracil) is a major selective agent in the treatment of colorectal cancer, but it can temporarily reduce the size of colon cancer, but the patient's survival is substantially prolonged or healed There is little evidence to suggest that. Also, despite the marginal effects of 5-FU, there were no other drugs or combination therapies that appeared to be more effective.

In general, substances used as anticancer drugs have considerable toxicity, and they do not selectively remove cancer cells, and cause fatal damage to normal cells, resulting in hemopoiesis such as leukocytes, platelets and red blood cells due to bone marrow destruction; Hair loss symptoms due to hair follicle cell destruction; Side effects of ovaries and testicles cause menstrual irregularities and male infertility; Side effects from digestive mucous membrane destruction include stomatitis, nausea and vomiting and food swallowing disorders and digestive disorders; Diarrhea symptoms; Renal toxicity by tubular necrosis; Peripheral neuritis and weakness caused by nervous system disorders; Vascular disorders such as vascular pain and rash; Skin and nail discoloration.

Therefore, it is urgently required to develop a chemotherapeutic agent having low toxicity and effective to prevent the development of cancer, as well as research to raise the therapeutic effect while minimizing side effects of the anticancer agent and treatment of cancer after the cancer. In addition, the biggest problem of chemotherapeutic agents is drug resistance, which is the main cause of failure in the end despite the initial successful response by the anticancer drug. Therefore, It is becoming important.

On the other hand, Treculia africana is one of foreign biological active plants which is also called African breadfruit. It is known that a genus Treculia plant has been used as an antimycobacteria, anti-reverse transcriptase, radical Although there are reports of scavenging and antitumor activity (South african journal of botany 76 (3): 530-535, 2010), there is no known anticancer activity against other biological activities, especially colon cancer .

Under these circumstances, the present inventors have made intensive studies to find natural products having anticancer effects and less side effects, and as a result, it has been found that the extract of Trekulia african, a kind of foreign bio-active plant as described above, can effectively kill cancer cells, especially colon cancer cells And completed the present invention.

It is an object of the present invention to provide a pharmaceutical composition for the prevention or treatment of colorectal cancer comprising extract of Treculia africana as an active ingredient.

Another object of the present invention is to provide a food composition for preventing or ameliorating colorectal cancer comprising Trekulia africana or extract as an active ingredient.

In one aspect of the present invention, the present invention provides a pharmaceutical composition for preventing or treating colorectal cancer comprising Treculia africana extract as an active ingredient.

The term " Treculia africana "of the present invention, also called African breadfruit, is a family Moraceae genus Treculia plant, one of the edible plants. The Trekulia african is one of the food crops containing a lot of starch, which is the fruit of the bread tree, also called artocarpus (Ulu). Originating in the African tropics, it is a specialty plant that grows in the Malay Islands. It is cultivated from ancient times and spread all over the South Pacific tropics and is also grown in South America, from Mexico to Brazil. The morphological and physiological ecological characteristics of the Trekulia africana are known, but the growth characteristics such as maturation, harvesting and cultivation of the fruits are known, but their activity is not well known, and the leaf extract of Trekulia Africa Although there is a report that it has tumor activity, it has antitumor activity only against Agrobacterium tumefaciens, a tumor cell line disease, but there is no known anticancer activity against colon cancer.

The term "extract " of the present invention means a preparation which is obtained by squeezing a herbal medicine with an appropriate leaching solution and concentrating by evaporating the leaching solution, and is not limited thereto. The extract, the diluted solution, A dried product obtained by drying, a controlled preparation thereof, or a purified product thereof. The extracts of Trekulia africana can be prepared using common extraction, isolation and purification methods known in the art. The extraction method may be, but not limited to, hot water extraction, hot water extraction, cold extraction, reflux cooling extraction, or ultrasonic extraction.

According to a specific embodiment of the present invention, the Trekulia africanus extract of the present invention is prepared by crushing a certain amount of Treukyuria africana powder into powder and extracting it with 5 to 10 times of a solvent. As the solvent, an organic solvent may be used, and the organic solvent may be water, C1 to C4 lower alcohol or a mixture thereof, preferably methanol. Further, the extraction can be preferably carried out at a temperature of 35 to 55 ° C, more preferably at 45 ° C. As described above, the extraction method can be carried out by using any method known in the art without limitation, but it can be preferably extracted by ultrasonication. The extraction was repeated several times, and the extracted sample was filtered, concentrated and lyophilized.

In the present invention, the term "prevention" means any action that inhibits or delays the development, spread and recurrence of colorectal cancer by the administration of the pharmaceutical composition according to the present invention. , Which means all suspicions of colorectal cancer and any behavior that alters or alleviates symptoms of the onset individuals.

Preferably, the prevention or treatment of colorectal cancer can be accomplished by the apoptosis of colon cancer cells induced by Trekulia africana or extract according to the present invention. The above-mentioned "apoptosis" is a kind of programmed cell death (PCD), in which abnormal cells, damaged cells and aged cells are killed and killed by the biological program inputted into our body, . Such apoptosis is distinguished from cell death caused by stimulation such as burns, scabs, and poisons, such as necrosis, which is the death or accidental death of a cell. That is, apoptosis is an active death of a cell that is regulated by a protein expressed from a specific gene. While apoptosis occurs chaotically, apoptosis occurs in an orderly fashion in a short time. Specifically, apoptosis begins with the contraction of the cell, creating a gap between the adjacent cells, and DNA, which is a genetic material in the nucleus of the cell in the process of dying, is regularly cleaved into fragments. Ultimately, the entire cell called the apoptotic body is also broken up, and it is killed by other cells around it. It is involved in the formation of the human body during the early development of the human body. In adults, it is responsible for eliminating normal cells when they become senescent, or removing cells with abnormalities. The apoptosis is distinguished from PCD in that it also occurs in cases other than PCD, and is related to cell death due to cancer cell death, viral infection, drug, and radiation.

According to a specific embodiment of the present invention, the composition of the present invention, for example, the extract of Trekulia africanum or methanol showed no cytotoxicity against normal cells (3T3-L1), but for cancer cell lines (A549, HT29 and HepG2) (A549) and hepatocellular carcinoma cells (HepG2), in particular, the cancerous cell line of the colon cancer cell (HT29) (FIG. 1). In addition, it was confirmed that the treatment of the composition effectively induced apoptosis of colon cancer cells by inducing apoptosis, thereby further demonstrating excellent anticancer activity (FIG. 2). Further, by confirming the expression of proteins associated with apoptosis of cancer cells, it was confirmed once again that the composition of the present invention induces apoptosis of cancer cells through an external pathway through a death receptor (FIG. 3).

The pharmaceutical composition of the present invention may further comprise a pharmaceutically acceptable carrier. The term "pharmaceutically acceptable" of the present invention means that it exhibits properties that are not toxic to the cells or humans exposed to the composition. Such carriers may be used without limitation as long as they are known in the art such as buffers, preservatives, wetting agents, solubilizers, isotonic agents, stabilizers, bases, excipients and lubricants.

In addition, the pharmaceutical composition of the present invention may be formulated in the form of powders, granules, tablets, capsules, suspensions, emulsions, syrups, aerosols and the like, oral preparations, suppositories and sterilized injection solutions according to a conventional method have. Furthermore, it can be used in the form of an external preparation for skin in the form of ointments, lotions, spray agents, patches, creams, powders, suspensions, gels or gels. Examples of carriers, excipients and diluents that can be included in the composition of the present invention include lactose, dextrose, sucrose, sorbitol, mannitol, xylitol, erythritol, maltitol, starch, acacia rubber, alginate, gelatin, calcium phosphate, calcium silicate, Cellulose, methylcellulose, microcrystalline cellulose, polyvinylpyrrolidone, water, methylhydroxybenzoate, propylhydroxybenzoate, talc, magnesium stearate and mineral oil. In the case of formulation, a diluent or excipient such as a filler, an extender, a binder, a wetting agent, a disintegrant, or a surfactant is usually used.

Solid formulations for oral administration include tablets, pills, powders, granules, capsules and the like, which may contain at least one excipient such as starch, calcium carbonate, Sucrose, lactose, gelatin and the like. In addition to simple excipients, lubricants such as magnesium stearate and talc are also used. Liquid preparations for oral use may include various excipients such as wetting agents, sweetening agents, fragrances, preservatives, etc. in addition to water and liquid paraffin, which are simple diluents commonly used in suspension, liquid solutions, emulsions and syrups have. Formulations for parenteral administration include sterilized aqueous solutions, non-aqueous solutions, suspensions, emulsions, freeze-dried preparations, and suppositories. Examples of the suspending agent include propylene glycol, polyethylene glycol, vegetable oil such as olive oil, injectable ester such as ethyl oleate, and the like. Examples of the suppository base include witepsol, macrogol, tween 61, cacao butter, laurin, glycerogelatin and the like.

Meanwhile, the pharmaceutical composition of the present invention is administered in a pharmaceutically effective amount. The term "pharmaceutically effective amount " of the present invention means an amount sufficient to treat a disease at a reasonable benefit / risk ratio applicable to medical treatment and not causing side effects, The type of cancer, the severity, the activity of the drug, the sensitivity to the drug, the method of administration, the time of administration, the route of administration and the rate of release, the duration of the treatment, factors including the drugs used concurrently or concurrently and other factors well known in the medical arts . The composition of the present invention may be administered as an individual therapeutic agent or in combination with other therapeutic agents, and may be administered sequentially or simultaneously with conventional therapeutic agents, and may be administered singly or multiply. It is important to take into account all of the above factors and to administer the amount in which the maximum effect can be obtained in a minimal amount without side effects, which can be easily determined by those skilled in the art.

Specifically, the effective amount of the extract contained in the composition of the present invention may vary depending on the age, sex, and body weight of the patient. In general, the Trekulia africanus extract of the present invention is 0.001 To 100 mg, preferably 1 to 10 mg / kg body weight per day, more preferably 1 to 5 mg per day or every other day or one to three divided doses per day. However, the dosage may not be limited in any way because it may be increased or decreased depending on route of administration, severity of disease, sex, weight, age, and the like.

The composition of the present invention may be administered to mammals such as rats, mice, livestock, humans, and the like in various routes. All modes of administration may be expected, for example, by oral, rectal or intravenous, intramuscular, subcutaneous, intra-uterine or intracerebroventricular injections.

In another aspect, the present invention provides a method of preventing or treating colorectal cancer comprising administering Trekulia africa or an extract to a subject in need thereof.

The term "individual" of the present invention means a mammal such as a monkey, a cow, a horse, a sheep, a pig, a chicken, a turkey, a quail, a cat, a dog, a mouse, a rat, a rabbit or a guinea pig , And the diseases can be effectively prevented or treated by administering the pharmaceutical composition of the present invention to an individual. The pharmaceutical composition of the present invention can be administered in parallel with existing therapeutic agents.

The term "administering" of the present invention means providing the patient with the desired substance in any suitable manner, and the administration route of the composition of the present invention may be administered through any conventional route so long as it can reach the target tissue have. But are not limited to, intraperitoneal, intravenous, intramuscular, subcutaneous, intradermal, oral, topical, intranasal, intrathecal, rectal. In addition, the pharmaceutical composition of the present invention may be administered by any device capable of moving the active substance to the target cell. The preferred modes of administration and formulations are intravenous, subcutaneous, intradermal, intramuscular, and drip injections. The injectable solution may be a non-aqueous solvent such as an aqueous solvent such as a physiological saline solution or a ring gel solution, a vegetable oil, a higher fatty acid ester (e.g., oleic acid), an alcohol (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.) (For example, ascorbic acid, sodium hydrogen sulfite, sodium pyrophosphate, BHA, tocopherol, EDTA and the like), an emulsifier, a buffer for pH control, a microbial growth inhibitor And a pharmaceutical carrier such as a preservative (e.g., mercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.).

The term "therapeutically effective amount " used in connection with the active ingredient in the present invention means the amount of a pharmaceutically acceptable salt of Trekulia africanus or an extract effective to prevent or treat the subject disease.

The pharmaceutical composition of the present invention may further comprise a known anticancer agent in addition to Treukyuria africanus or extract as an active ingredient and may be used in combination with other therapies known for the treatment of these diseases. Other treatments include, but are not limited to, chemotherapy, radiation therapy, hormone therapy, bone marrow transplantation, stem cell replacement therapy, other biological therapies, immunotherapy, and the like.

In another aspect, the present invention provides a food composition for preventing or ameliorating colorectal cancer comprising Trekulia africana or an extract as an active ingredient. When the composition of the present invention is used as a food composition, the Trekulia african and its extracts can be directly added or used together with other food or food ingredients, and can be suitably used according to a conventional method. The composition may contain a food-acceptable food-aid additive in addition to the active ingredient, and the amount of the active ingredient to be mixed may be suitably determined according to the purpose of use (prevention, health or therapeutic treatment).

As used herein, the term "food-aid additive " refers to a component that can be added to foods in a supplementary manner, and is appropriately selected and used by those skilled in the art as added to produce health functional foods of each formulation. Examples of food-aid additives include flavors such as various nutrients, vitamins, minerals (electrolytes), synthetic flavors and natural flavors, colorants and fillers, pectic acid and its salts, alginic acid and its salts, organic acids, , a pH adjusting agent, a stabilizer, a preservative, a glycerin, an alcohol, and a carbonating agent used in a carbonated drink. However, the types of the food auxiliary additives of the present invention are not limited by these examples.

A health functional food may be included in the food composition of the present invention. The term "health functional food " as used in the present invention refers to a food prepared and processed in the form of tablets, capsules, powders, granules, liquids and rings using raw materials and components having useful functions in the human body. Here, 'functional' refers to the structure and function of the human body to obtain nutritional effects and obtain useful effects for health use such as physiological action. The health functional food of the present invention can be prepared by a method commonly used in the art and can be prepared by adding raw materials and ingredients that are conventionally added in the art. In addition, the formulations of the above health functional foods may also be manufactured without limitations as long as they are acceptable as health functional foods. The composition for food of the present invention can be manufactured in various forms, and unlike general pharmaceuticals, it has the advantage that there is no side effect that may occur when a drug is used for a long period of time, and is excellent in portability, Can be ingested as an adjuvant to enhance the effectiveness of anticancer drugs.

There is no limitation on the kind of health food to which the composition of the present invention can be used. In addition, the composition comprising the extract of Trekulia africana or extract of the present invention as an active ingredient may be prepared by mixing other suitable auxiliary ingredients and known additives, which may be contained in health functional foods, according to the selection of a person skilled in the art. Examples of foods that can be added include dairy products, such as meat, sausage, bread, chocolates, candies, snacks, confectionery, pizza, ramen, other noodles, gums, ice cream, various soups, drinks, tea, Vitamin complex, and the like, and can be prepared by adding to the juice, tea, jelly, and juice prepared from the extract of the present invention as a main component.

The extract of Trekulia africanus according to the present invention can be used as a pharmaceutical composition or a food composition because natural plant is used as a raw material, so that side effects can be less than general synthetic compounds. Therefore, it can be safely contained in a pharmaceutical composition and a health functional food, Can be used.

The composition comprising the extract of Trekulia africanus extract of the present invention does not show toxicity to normal cells but induces apoptosis of colon cancer cells and exhibits remarkably excellent colon cancer cell killing effect and thus can be usefully used as an anticancer composition , A pharmaceutical composition or a food composition for the prevention or treatment of colon cancer.

FIG. 1 is a graph showing the cancer cell killing effect of a methanol extract of Treculia africana (META) of a foreign biological active plant Trekulia africana according to the present invention.
FIG. 2 is a graph showing the cell death-inducing effect of META according to the present invention on colon cancer cell HT29. (A) is a graph showing the results of flow cytometry on cells stained with FITC-Annexin V and PI (propidium iodide) after META treatment, and (B) .
FIG. 3 is a graph showing the ability of META according to the present invention to regulate the expression of an apoptosis-related protein against colorectal cancer cell HT29.

Hereinafter, the constitution and effects of the present invention will be described in more detail through examples. These examples are only for illustrating the present invention, and the scope of the present invention is not limited by these examples.

Example 1: Trachelia Africa ( Treculia africana ) Preparation of extract

Treculia africana Decne., As a foreign bio-active plant, was sold through the Korea Plant Extract Bank of the Overseas Biomaterials Hub Center, Korea Research Institute of Bioscience and Biotechnology. The plant samples were put into 99.9% methanol and sonicated at 45 ° C with a sonicator to extract. The extract was concentrated, lyophilized and stored. In the experiment, lyophilized samples were dissolved in dimethyl sulfoxide (DMSO).

Example 2: Culture of cell lines

In order to confirm the cytotoxicity of the extract of Treculia africana (META) produced in Example 1 and the methanol extract of Treculia africana (META) prepared in Example 1 and to confirm the specific killing effect on colon cancer cells, Together, normal cell line 3T3-L1, lung cancer cell line A549, and hepatoma cell line HepG2 were purchased from the American Type Culture Collection (ATCC). A549, HT29 and HepG2 were cultured in DMEM (Dulbecco's modified Eagle's medium) medium containing 10% fetal bovine serum (FBS) and penicillin / streptomycin, and 3T3-L1 was cultured in 10% calf And cultured in DMEM medium containing serum (bovine calf serum; BCS) and penicillin / streptomycin.

Example 3 Analysis of Colon Cancer Cell Killing Effect of Trekulia africana and Extracts

The safety of META prepared in Example 1 on normal cells and the specific killing effect on colon cancer cell lines were confirmed. Cancer cell lines and normal cell lines were seeded and cultured in a 24-well tissue culture dish at a density of 1 × 10 ⁴ to 3 × 10 ⁴ cells per well and treated with various concentrations of META (0 to 200 μg / ml) for 48 hours After incubation, cell death induction was analyzed by WST assay. The results are shown in FIG. 1 and Table 1.

As shown in FIG. 1, it was confirmed that META does not show toxicity to the normal cell line (3T3-L1), but basically has a killing effect on cancer cells (A549, HT29 and HepG2). Among them, it was confirmed that the colon cancer cell line (HT29) had a much better killing effect than the lung cancer cell line (A549) and hepatocarcinoma cell line (HepG2). In addition, the concentration showing 50% killing effect on each cancer cell line was calculated, and as shown in Table 1, 62.1 占 퐂 / ml for colon cancer cell line (HT29) was found to be superior to other cancer cell lines I could confirm.

Methanol extract IC ₅₀ ([mu] g / ml) A549 HT29 HepG2 META 221.9 62.1 215.9

Example 4 Analysis of Apoptosis Inducibility of Trekulia africana and Extracts

In order to elucidate the mechanism of the anticancer activity of META prepared in Example 1, the ability of the extract to induce apoptosis was confirmed. Particularly, when apoptosis is induced, phosphatidylserine, a component of the cell membrane, is exposed to the outside of the cell from inside the cell. Thus, the degree of apoptosis was confirmed using Annexin V, which specifically binds to phosphatidylserine. Specifically, colon cancer cell lines were seeded and cultured in a 6-well tissue culture dish at a rate of 1 × 10 ⁵ to 3 × 10 ⁵ cells per well. META was treated at various concentrations for 48 hours, cells were recovered, And stained with Annexin V & Dead Cell Kit (Merck Millipore, Darmstadt, Germany). The stained cells were analyzed using a flow cytometer (Muse ™ Cell Analyzer, Merck Millipore, Darmstadt, Germany) and the results are shown in FIG.

As shown in FIG. 2, when META was treated at different concentrations of CT29 for colorectal cancer cells, the proportion of living cells decreased in a concentration-dependent manner and the proportion of early apoptotic cells and late apoptotic cells increased, The proportion of apoptotic cells was rapidly increased. That is, it was confirmed that apoptosis was increased depending on the treatment concentration of META.

Example 5: Analysis of anticancer activity-related protein expression control activity of Trekulia africana extract

To elucidate the mechanism of the anticancer activity of META prepared in Example 1, expression levels of p53, p21 and caspase, which are apoptosis-related proteins of the extract, were analyzed by Western blot hybridization. Specifically, the protein was extracted from the cancer cell line treated with META as described above, and the protein concentration was determined using a Bradford assay. Then, 30 μg of the protein was electrophoresed on 10% SDS-PAGE, blotted onto nitrocellulose membrane, And then hybridized with the antibody to the antibody. After the membrane was washed, horseradish peroxidase (HRP) was reacted with the tagged secondary antibody for 1 hour and analyzed using a chemiluminescence detection system, as shown in FIG.

As shown in FIG. 3, when META was administered to human colon cancer cell HT29, the expression of Fas, a death receptor, was increased. Furthermore, the caspase cascade was activated to activate caspase 8 and caspase 3 The expression of the active caspase-8 and caspase-3 was increased. In addition, it was confirmed that PARP (poly ADP-ribose polymerase) fragment involved in DNA repair and gene transcription, which is one of substrate proteins, is activated by activated caspase 3. In addition, the expression level of p53, a tumor suppressor gene, was not changed by the treatment with META, but the expression level of p21, which is a CDK inhibitor, was increased. These results confirm that META induces apoptosis of cancer cells by external pathway through death receptor.

In general, the extract of Trekulia africana according to the present invention shows a killing effect specifically on cancer cells without showing toxicity to normal cells, and exhibits a remarkably excellent killing effect on colon cancer cells, The extracts showed anticancer effects by inducing apoptosis of colon cancer cells.

Claims (5)

A pharmaceutical composition for preventing or treating colorectal cancer comprising extract of Treculia africana as an active ingredient.
The method according to claim 1,
Wherein the extract is extracted with water, C1 to C4 lower alcohol or a mixed solvent thereof.
The method according to claim 1,
Wherein the alcohol is methanol.
The method according to claim 1,
Wherein the prevention or treatment of colon cancer is achieved by inducing apoptosis of cancer cells.
A food composition for preventing or ameliorating colorectal cancer, comprising extract of Trekulia africana or an extract as an active ingredient.

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